Monitoring conformational dynamics of a single molecule by selective fluorescence spectroscopy.
A recently developed, real-time spectroscopic technique, burst-integrated fluorescence lifetime (BIFL), is shown to be well suited for monitoring the individual molecular conformational dynamics of a single molecule diffusing through the microscopic, open measurement volume (approximately 10 fl) of a confocal epi-illuminated set-up. In a highly diluted aqueous solution of 20-mer oligonucleotide strand of DNA duplex labeled with the environment-sensitive fluorescent dye tetramethylrhodamine (TMR), fluorescence bursts indicating traces of individual molecules are registered and further subjected to selective burst analysis. The two-dimensional BIFL data allow the identification and detection of different temporally resolved conformational states. A complementary autocorrelation analysis was performed on the time-dependent fluctuations in fluorescence lifetime and intensity. The consistent results strongly support the hypothesized three-state model of the conformational dynamics of the TMR-DNA duplex with a polar, a nonpolar, and a quenching environment of TMR.
Eggeling
C
C
Max-Planck-Institut für Biophysikalische Chemie, Am Fassberg 11, D-37077 Göttingen, Germany.
Fries
J R
JR
Brand
L
L
Günther
R
R
Seidel
C A
CA
D007700:Kinetics; D009038:Motion; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides; D012235:Rhodamines; D013050:Spectrometry, Fluorescence / Q000379:methods*
D009838:Oligodeoxyribonucleotides; D012235:Rhodamines